This invention provides a fibrin porous structure material and particularly a fibrin porous structure having good resistance to compression and a porosity formed by large cells.
Fibrin sponges are known from U.S. Pat. No. 4,442,655 and WO99/15209. The fibrin sponges have a porous structure and have substantially no compression strength. After hydration, the sponges have cells having a cross section area of less than about 100 xcexcm2, i.e., a porosity which is different from the porosity of a natural human bone.
As explained in the ""655 patent, the sponge structure is obtained by freeze-drying a reaction mixture containing fibrinogen, fibrin and a catalytic amount of thrombin. None of the examples of the ""655 patent relates to the freeze-drying of a solution containing fibrin partially cross-linked due to the presence of an anticoagulant increasing the clotting time.
It has now been found that, after lyophilizing a solution containing fibrin partially cross-linked due to the presence of a sufficient amount of a calcium inhibiting or blocking agent, preferably an anticoagulant, it was possible to obtain a solid porous structure having an open cross section of more than 500 xcexcm2 and/or a wall thickness between channels of at least 10 xcexcm. Those skilled in the art were unable to predict that it would be able to prepare fibrin porous structure having a good resistance to compression and a porosity formed by large cells, such as in the natural human bone.
The invention relates to a porous structure comprising fibrin or fibrinogen material, and possibly other compounds, the structure having:
in its substantially dry form, a compression strain of less than about 8%, preferably less than about 7%, and most preferably from about 6 and to about 7%, and a creep modulus higher than 1.5xc3x97106 Pa, advantageously higher than 1.7xc3x97106 Pa, most preferably from about 1.8xc3x97106 Pa to about 2.5xc3x97106 Pa, said compression strain and creep modulus being measured for a sample having a diameter of 5 mm on which a compression of 2500 milli Newtons is exerted with a compression ramp of 500 milli Newtons per minute, after a compression release step following an initial compression of 2500 milli Newtons with a compression ramp of 500 milli Newtons per minute, and
after hydration, such a porosity that at least about 50% by volume of the total porosity is formed by channels with an open cross section of more than 500 xcexcm2.
These large pores are suitable for the attachment of cells on the structure. Moreover, according to an embodiment, such large pores are similar to the pores of human natural bones.
Advantageously, the mechanical properties of the porous structure of the invention are kept after successive compression steps, separated the one from another by a release step. Advantageously, the structure, in its substantially dry form, has a compression strain of less than about 8%, more preferably less than about 7%, most preferably from about 6% to about 7%, and a creep modulus higher than 1.5xc3x97106 Pa, advantageously higher than 1.7xc3x97106 Pa, for example between about 1.8xc3x97106 Pa to about 2.5xc3x97106 Pa, the compression strain and creep modulus being measured for a sample having a diameter of 5 mm on which a compression of 2500 milli Newtons is exerted with a compression ramp of 500 milli Newtons per minute, after ten cycles consisting of a compression step of 2500 milli Newtons with a compression ramp of 500 milli Newtons per minute followed by a compression release step.
It has also been observed that it was interesting to adjust the atomic ratio of calcium and phosphorus (Ca/P) of the structure, preferably between 1 and 2, most preferably between 1.67 and 1.95, for the formation of hydroxyapatite.
The structure of the invention can be used, after grinding, as a powder. Such a powder can, for example, be added to a liquid or to form a liquid glue.
The structure of the invention that has a good compression resistance and larger pores can be used as support for a skin layer and/or for a sponge structure, especially a fibrin sponge structure.
A titanium support can also be provided with a layer of the porous structure of the invention, or with a layer of a powder of the porous structure of the invention, or with a layer containing a powder of the structure of the invention. Preparation processes of such titanium supports will be disclosed after the description of processes of preparation of the structure of the invention.
The present invention further provides a process for preparing a porous structure of the invention. The process provides the steps of (1) providing a solution containing fibrin or fibrinogen materials (advantageously at least 3 mg/ml), (2) polymerizing the fibrin or fibrinogen, preferably a polymerization with at least partial cross-linking of the fibrin or fibrinogen materials in the presence of a calcium blocking or inhibiting agent (preferably an anticoagulant), and (3) lyophilizing the polymerized fibrin or fibrinogen. The calcium blocking or inhibiting agent should be present in the solution in an amount sufficient for preparing a porous structure. The resulting fibrin or fibrinogen material should have:
in its substantially dry form, a compression strain of less than about 8%, preferably less than about 7%, and more preferably from about 6% to about 7%, and a creep modulus higher than 1.5xc3x97106 Pa, more preferably higher than 1.7xc3x97106 Pa, most preferably from about 1.8xc3x97106 Pa to about 2.5xc3x97106 Pa, the compression strain and creep modulus being measured for a sample having a diameter of 5 mm on which a compression of 2500 milli Newtons is exerted with a compression ramp of 500 milli Newtons per minute, after a compression release step following an initial compression of 2500 milli Newtons with a compression ramp of 500 milli Newtons per minute, and
after hydration, such a porosity that at least 50% by volume of the total porosity is formed by channels with an open cross section of more than about 500 xcexcm2.
Calcium inhibiting agent means an agent suitable for inhibiting at least partly the functionality of one or more calcium sites of the fibrin or fibrinogen material. The calcium inhibiting agent is preferably a calcium blocking agent, most preferably an anticoagulant.
The present invention relates to a porous structure comprising fibrin or fibrinogen material, and possibly other compounds, the structure having:
in its substantially dry form, a compression strain of less than about 8%, preferably less than about 7%, and more preferably from about 6% to about 7%, and a creep modulus higher than 1.5xc3x97106 Pa, more preferably higher than 1.7xc3x97106 Pa, most preferably from about 1.8xc3x97106 Pa to about 2.5xc3x97106 Pa, the compression strain and creep modulus being measured for a sample having a diameter of 5 mm on which a compression of 2500 milli Newtons is exerted with a compression ramp of 500 milli Newtons per minute, after a compression release step following an initial compression of 2500 milli Newtons with a compression ramp of 500 milli Newtons per minute, and
after hydration, such a porosity that at least 50% by volume of the total porosity is formed by channels with an open cross section of more than about 500 xcexcm2.
In the present specification, the wordings xe2x80x9csubstantially dry formxe2x80x9d mean a residual moisture level of less than about 1%, and more preferably of less than about 0.5%.
The structure of the invention has advantageously a low moisture content, for example a moisture content of less than about 7.5%, preferably of less than about 2%, most preferably of less than about 1%, and more specifically less than about 0.5%. In the event the moisture content of the structure of the invention is greater than 1%, for the determination of the mechanical properties of the structure of the invention in its substantially dry form, the moisture content has to be lowered to less than about 1%, by using a technique not degrading the bounds of the structure.
In a preferred form of the invention, the mechanical properties of the porous structure of the invention are retained after successive steps of compressing the structure with release steps between the compressing steps. Advantageously, the structure, in its substantially dry form, has a compression strain of less than about 8%, preferably less than about 7%, and more preferably from about 6% to about 7%, and a creep modulus higher than 1.5xc3x97106 Pa, more preferably higher than 1.7xc3x97106 Pa, most preferably from about 1.8xc3x97106 Pa to about 2.5xc3x97106 Pa, the compression strain and creep modulus being measured for a sample having a diameter of 5 mm on which a compression of 2500 milli Newtons is exerted with a compression ramp of 500 milli Newtons per minute, after ten cycles consisting of a compression step of 2500 milli Newtons with a compression ramp of 500 milli Newtons per minute followed by a compression release step.
Preferably, the mechanical properties of the porous structure of the invention are at least retained in part after rehydration. Advantageously, the structure, in its hydrated form, has a compression strain of less than about 8%, preferably less than about 7%, and more preferably from about 6% to about 7%, and a creep modulus higher than 1.5xc3x97106 Pa, more preferably higher than 1.7xc3x97106 Pa, most preferably from about 1.8xc3x97106 Pa to about 2.5xc3x97106 Pa, the compression strain and creep modulus being measured for a sample having a diameter of 5 mm on which a compression of 2500 milli Newtons is exerted with a compression ramp of 500 milli Newtons per minute, after one cycle, preferably after ten cycles, consisting each of a compression step of 2500 milli Newtons with a compression ramp of 500 milli Newtons per minute followed by a compression release step.
Advantageously, the structure has fibrin or fibrinogen dimensions such that, after hydration, the ratio of the volume of hydrated structure versus the volume of the dry structure is from about 0.5 to about 1.5, more preferably from about 0.7 to about 1.2, and most preferably from about 0.9 to about 1.1.
According to a preferred embodiment, the structure comprises walls defining therebetween cells, at least part of said cells are linked so as to define channels having after hydration in cross section an open section greater than about 500 xcexcm2, more preferably greater than about 1000 xcexcm2, and most preferably from about 3,000 xcexcm2 to about 300,000 xcexcm2. Preferably, at least 50% of the total porosity of the structure is formed by channels with an open cross section of more than about 1000 xcexcm2. According to a detail of a specific embodiment, the channels of the structure are homogeneously dispersed and have a substantially identical open cross-section. For example, 80% of the total porosity is formed by channels with an open cross-section from about 0.75 to about 2 times the mean open cross section of said channels. In order to determine the open cross section of the channels of the structure, the structure is cut perpendicular or substantially perpendicular to the axis of the channels, said cut being thereafter visualized by means of a microscope, electronic microscope, or any appropriate techniques.
According to an embodiment, the structure has an apparent density calculated in its substantially dry form of less than about 0.5 g/cm3, advantageously less than about 0.3 g/cm3, for example between 0.05 and 0.2 g/cm3. Apparent density corresponds to the weight of a cube of 1 cm3 of the porous structure. When the channels of the porous structure are filled with water by immersing the structure into a bath, the structure falls towards the bottom of the bath. By determining the volume of the walls of the structure (volume increase of the bath when immersing a cube of 1 cm3 of the porous structure in the bath) and by determining the weight of the cube, it is possible to determine the density of the walls of the structure.
The walls have advantageously an average thickness of less than about 100 xcexcm, advantageously from about 10 to about 80 xcexcm, preferably from about 20 to about 60 xcexcm. The thickness is for example measured by scanning electron microscope (SEMxe2x80x94Philips XL20)
As the channels of the structure have a high open cross section, the surface area of the structure is low. For example, the surface area of the structure is less than about 1 m2/g, especially less than about 0.5 m2/g.
Preferably, the atomic ratio Ca/P of the structure is adjusted. For example, the atomic ratio is from about 0.5 to about 5, preferably lower than about 2, most preferably from about 1.67 to about 1.95.
According to an embodiment, the structure contains calcium which is substantially not bound to albumin and/or to fibrin and/or to fibrinogen materials, and/or the structure has a low albumin content, for example the weight ratio fibrin/albumin of the solution used for the preparation of the structure is greater than about 2, advantageously greater than about 4, most preferably greater than about 8.
The structure has, for example, a ratio of the volume of the porous structure in its substantially dry form to the volume of the structure in its hydrated form (i.e. hydrated after the lyophilization step) is from about 0.7 to about 1.7, advantageously from about 0.8 to about 1.2, and most preferably about 1.
The structure can be sterilized, by treatment with sterilizing agent, by gamma radiation, by X rays, with ethylene oxide, etc. The treatment is preferably a treatment that does not denature the fibrin. Sterilization of the structure is preferably below xe2x88x9225xc2x0 C., and most preferably below xe2x88x9280xc2x0 C. at a dosage of at least 25 kGy.
The structure of the present invention is substantially pyrogen free.
The structure of the present invention which is provided with cells or channels, comprises at least a binding agent, for example in the form of a layer, on at least a part of the surface of cells.
The structure of the present invention can also include an additive (for example within the wall of the channels or cells of the structure) or is provided with a layer containing at least an additive, said additive being selected from the group consisting of processing aids (such as lubricant, plastifying agent, surfactant, viscosity reducing agent, etc.), fibers, polymers, copolymers, antibody, antimicrobial agent, agent for improving the biocompatibility of the structure, proteins, anticoagulants, anti-inflammatory compounds, compounds reducing graft rejection, living cells, cell growth inhibitors, agents stimulating endothelial cells, antibiotics, antiseptics, analgesics, antineoplastics, polypeptides, protease inhibitors, vitamins, cytokine, cytotoxins, minerals, interferon""s, hormones, polysaccharides, genetic materials, proteins promoting or stimulating the growth and/or attachment of endothelial cells on the cross-linked fibrin, growth factors,.cell growth factors, growth factors for heparin bond, substances against cholesterol, pain killers, collagen, osteoblasts, chondroblasts, chondrocytes, osteoclasts, hematpoeitic cells, stromal cells, osteoprogenitor cells, drugs, anti coagulants, poly DL lactate, alginate, recombinant material, triglycerides, fatty acids, C12-C24 fatty acids, etc. and mixtures thereof.
The term xe2x80x9cgenetic materialxe2x80x9d as used herein refers to nucleotide based materials, including without limitation, viruses and viral fragments, deoxyribonucleic acid (DNA), plasmids, ribonucleic acid (RNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), catalytic RNA (cRNA), smaller nuclear RNA (snRNA), exons, introns, codons, and anti-sense oligonucleotides. Genetic material, especially viruses and viral fragments, may incidentally include some protein. In addition, the term xe2x80x9crecombinant materialxe2x80x9d as used herein refers to material manufactured by recombinant technology which is a series of procedures that are used to join (recombine) segments of two or more different DNA molecules. A recombinant DNA molecule can enter a cell and replicate there, autonomously, or after it has become integrated into a chromosome.
The structure of the present invention comprises, according to a preferred embodiment, bone chips, such as bone chips or particles having an average particle size (average by weight) lower than 2 mm, for example from about 100 m to about 1 mm, advantageously from about 250 xcexcm to about 750 xcexcm, and most preferably about 500 xcexcm.
The structure of the present invention contains for example from 1 to 85% by weight a bone chips, advantageously from 5 to 60%, preferably from 10 to 50%, most preferably from 15 to 30% by weight bone chips.
Another object of the invention is a powder of the structure of the invention. Such a powder can be prepared by milling or grinding a structure of the invention. The grain size of the powder is for example lower than 5 mm, advantageously from about 500 xcexcm to about 2 mm. The grain size is however preferably lower than 1 mm.
Such a powder is possibly mixed with other solid particles, advantageously with bone chips, such as bone chips or particles having an average particle size (average by weight) lower than 2 mm, for example from about 100 xcexcm to 1 mm, advantageously from about 250 xcexcm to 750 xcexcm, preferably about 500 xcexcm.
Such a powder, advantageously mixed with bone particles, is suitable for preparing a glue or cement, for example for filling a hole in a bone. Advantageously, the glue is in a substantially liquid form or in the form of a paste, so that its application is easy. The powder of the invention, which possibly contains other additive(s), is thus used in the glue as filler. The glue or cement used can be any suitable and compatible glue or cement, said glue or cement possibly containing additive(s), active agent(s), etc.
A further object of the invention is a product consisting of powder of the invention which have been pressed together, possibly in presence of an additive, such as a binding agent, a lubricant, a plastizer, etc. and/or in presence of bone chips. The product has a higher density, is compact and the pores have a reduced pore size, for example an average pore size of less than 50 xcexcm, advantageously less than 20 xcexcm, preferably less than 10 xcexcm. The product is characterized by a high volume increase when hydrated.
Still a further product of the invention consists of a laminated product comprising at least a layer having the structure of the invention. For example, the product comprises several layers having a structure of the invention or a layer with the structure of the invention forming an intermediate layer extending between two layers. The laminated product can possibly be compacted. The laminated product is dense, compact and has a high resistance to compression.
A further object of the invention is a bone substitute made of a structure according to the invention. The bone substitute can be manufactured for example by mechanically working a block having a structure according to the invention, by filling a mold with a glue or cement containing particles or powders having a structure of the invention. Advantageously, the bone substitute is made of a product having a structure of the invention, the volume of which remains substantially constant when hydrated. Preferably, the bone substitute is characterized in that the difference between the volume of the bone substitute in its substantially dry form and the volume of the bone substitute in its hydrated form is less than +/xe2x88x925% of the volume.
Still a further object of the invention is a multilayer structure, said multi-layer structure comprising at least a structure of the invention. The multi-layer structure of the invention can for example comprise a first layer having a structure according to the invention, and a second layer selected from the group consisting of a structure according to the invention which is different from the first layer, a skin layer, a porous layer, a sponge layer, preferably the second layer is also a fibrin or fibrinogen layer.
The present invention relates also to a process for the preparation of a structure according to the invention. In said process, a solution containing fibrin or fibrinogen materials is polymerized, advantageously a polymerization with a at least partial cross-linking, of the fibrin or fibrinogen materials in presence of a calcium blocking or inhibiting agent (i.e. an agent inhibiting, advantageously blocking, the functionality of the calcium on one or more sites of the fibrin or fibrinogen material), and the solution of partially cross-linked fibrin or fibrinogen is lyophilized, whereby the calcium inhibiting or blocking agent is present in an amount sufficient for obtaining after the lyophilization a porous structure. The results structure having:
in its substantially dry form, a compression strain of less than about 8%, preferably less than about 7%, and more preferably from about 6% to about 7%, and a creep modulus higher than 1.5xc3x97106 Pa, more preferably higher than 1.7xc3x97106 Pa, most preferably from about 1.8xc3x97106 Pa to about 2.5xc3x97106 Pa, the compression strain and creep modulus being measured for a sample having a diameter of 5 mm on which a compression of 2500 Milli Newtons is exerted with a compression ramp of 500 Milli Newtons per minute, after a compression release step following an initial compression of 2500 Milli Newtons with a compression ramp of 500 Milli Newtons per minute, and
after hydration, such a porosity that at least 50% by volume of the total porosity is formed by channels with an open cross section of more than about 500 xcexcm2.
Advantageously, the calcium inhibiting or blocking agent is present in an amount sufficient for obtaining, after the lyophilization, a porous structure having mechanical properties which are substantially kept after successive compression steps, separated the one from another by a release step. Advantageously, the structure, in its substantially dry form, prepared by the process of the invention has a compression strain of less than about 8%, preferably less than about 7%, and more preferably from about 6% to about 7%, and a creep modulus higher than 1.5xc3x97106 Pa, more preferably higher than 1.7xc3x97106 Pa, most preferably from about 1.8xc3x97106 Pa to about 2.5xc3x97106 Pa, the compression strain and creep modulus being measured for a sample having a diameter of 5 mm on which a compression of 2500 milli Newtons is exerted with a compression ramp of 500 milli Newtons per minute, after ten cycles consisting of a compression step of 2500 milli Newtons with a compression ramp of 500 milli Newtons per minute followed by a compression release step.
Preferably, the calcium inhibiting or blocking agent is present in an amount sufficient for obtaining after the lyophilization a porous structure having mechanical properties which are substantially kept after rehydration. Advantageously, the structure prepared by a preferred process of the invention has, in its hydrated form, a compression strain of less than about 8%, preferably less than about 7%, and more preferably from about 6% to about 7%, and a creep modulus higher than 1.5xc3x97106 Pa, more preferably higher than 1.7xc3x97106 Pa, most preferably from about 1.8xc3x97106 Pa to about 2.5xc3x97106 Pa, the compression strain and creep modulus being measured for a sample having a diameter of 5 mm on which a compression of 2500 milli Newtons is exerted with a compression ramp of 500 milli Newtons per minute, after one cycle, preferably after ten cycles, consisting each of a compression step of 2500 milli Newtons with a compression ramp of 500 milli Newtons per minute followed by a compression release step.
Advantageously, the polymerization, with an at least partial cross-linking, of the fibrin or fibrinogen materials is carried out in presence of an amount of calcium inhibiting or blocking agent sufficient for inhibiting or reducing the cross-linking rate so as to obtain after lyophilization walls defining therebetween channels, said channels having after rehydration in cross section an open section greater than about 1000 xcexcm2, and most preferably from about 3,000 xcexcm2 to about 300,000 xcexcm2.
Advantageously, the polymerization, with an at least partial cross-linking, of the fibrin or fibrinogen materials is carried out in presence of an amount of calcium inhibiting agent, preferably a calcium blocking agent, most preferably an anticoagulant, that is sufficient for obtaining after lyophilization a wall thickness of less than 30 xcexcm, preferably of 5 to 15 xcexcm.
According to an embodiment, the calcium inhibiting or blocking agent is selected from the group consisting of: citrate salts, phosphate salts, oxalate salts and mixtures thereof. Other possible calcium blocking agents are compounds blocking calcium sites of the fibrinogen, such as sodium polyphosphate, zeolithe, phosphate, potassium citrate, salt of phosphonic acid, amine, glycol, diethyleneglycol, triethanolamine, ammonium, betaine, glycerophosphate, aluminosilicate (zeolithe P), hexametaphosphate, polyacrylate, oligomeric phosphates, polyethyleneglycol, tannic acid, verapamil salt, piperidine dion derivatives, guanidine derivatives, amlodipine benzenesulfonate, 3-methylflavone-8-carboxylic acid esters, compounds having antagonist properties towards calcium ion, lidoflazine, etc., anticoagulants such as dextran, 2,6 dimethyl 4(2nitrophenyl) 1,4 dihydro pyridine 3,5 dimethyl dicarboxylate (Nifedipine), heparin, ((diphenylacetyl-4,5 oxazolyl-2)immino)-2,2xe2x80x2 diethanol, trombarin, bisobrine, morphine, amphetamine, antibiotics having anti coagulant properties, sodium salt of the 4 amino 2 hydroxybenzoic acid, sodium citrate and mixtures thereof.
Preferably, the calcium blocking agent is also an anticoagulant such as dextran, 2,6 dimethyl 4(2nitrophenyl) 1,4 dihydro pyridine 3,5 dimethyl dicarboxylate (Nifedipine), heparin, ((diphenylacetyl-4,5 oxazolyl-2)immino)-2,2xe2x80x2diethanol, trombarin, bisobrine, morphine, amphetamine, antibiotics having anti coagulant properties, sodium salt of the 4 amino 2 hydroxybenzoic acid, sodium citrate and mixtures thereof.
The partial or complete polymerization, advantageously with a partial cross-linking, of the fibrin or fibrinogen is advantageously carried out at a pH from about 6 to about 10, more preferably from about 7 to about 8, most specifically at about 7.5. In order to ensure a substantially constant pH during the cross-linking, one or more buffers can be used. Preferably, the buffer has also a calcium inhibiting action.
Possibly, the partial or complete polymerization, advantageously with a partial cross-linking, is carried out at a pH lower than 6, but preferably at a pH sufficient for avoiding the denaturation of the fibrin or fibrinogen material. For example, the polymerization is carried out at least partly at a pH of from 4 to about 6, and preferably from about 5 to about 5.5. In this case, the calcium inhibiting or blocking agent is an acid, advantageously an organic acid, for example organic acid with 1 to 24 carbon atoms, most specifically citric acid, acetic acid, formic acid, tannic acid, etc., citric acid being preferred.
According to a possible embodiment, the polymerization is carried out in a first step at a first pH lower than about 6.5, and in a second step at a second pH higher than the first pH. For example, in the second step, the polymerization is carried out at a pH from about 6 to about 10, advantageously from about 6.5 to about 8, preferably at a pH from about 7 to about 7.5, and most specifically at a pH of from about 7.1 to about 7.4.
The polymerization, with, preferably, partial cross-linking, is carried out for example at a temperature from about 0xc2x0 C. to about 60xc2x0 C., advantageously at a temperature from about 5xc2x0 C. to about 50xc2x0 C., preferably at a temperature from about 20xc2x0 C. to about 40xc2x0 C., most preferably at a temperature of about 37xc2x0 C. The polymerization can possibly be carried out at various temperatures. For example, in a first step the polymerization is carried out at a first temperature, while in a second step, the polymerization is carried out at a temperature higher than the first temperature. For example, in the first step, the temperature of the reaction medium is lower than about 30xc2x0 C., preferably lower than about 25xc2x0 C., for example from about 15xc2x0 C. to about 25xc2x0 C., while in the second step, the temperature of the reaction medium is higher than 30xc2x0 C., for example from about 35xc2x0 C. to about 50xc2x0 C., most preferably about 37xc2x0 C. Possibly, the temperature of the reaction medium is increased during the reaction, for example according to a substantially continuous path. For example, the reaction starts at a temperature lower than or about 20xc2x0 C., and is progressively carried out at a temperature higher than 20xc2x0 C., for example at an end temperature of about 37xc2x0 C.
The fibrin or fibrinogen material after polymerization with partial cross-linking has advantageously, before its lyophilization, an osmolarity greater than about 175 mosm, preferably greater than about 200 mosm, most preferably greater than about 250 mosm, for example from about 250 to about 400 mosm. The fibrin or fibrinogen material after cross-linking has advantageously, before its lyophilization, has also advantageously an optical density lower than about 1 Absorbance Unit Full Scale, advantageously lower than about 0.5 AUFS. The osmolarity has been measured by measured by using the apparatus FISKE 2400 OSMOMETER (Fiske Associates) according to the following method:
A sample is supercooled several degrees below the freezing point. The heat of fusion liberated allows sample temperature to rise to a temporary xe2x80x9cliquid-solidxe2x80x9d equilibrium. The equilibrium is by definition the freezing point of the solution. The freezing point is related to stols to allow determination of osmolarity. The osmolarity is equal to the osmoles of solute per Kg of pure solvent.
The optical density has been measured at a wavelength of 800 nm (Absorbance Unit Full Scale).
According to a detail of a process of the invention, the polymerization, advantageously with an at least partial cross-linking of the fibrin or fibrinogen materials is carried out in presence of an effective amount of calcium inhibiting or blocking agent, preferably an anticoagulant, sufficient for having a clotting time of more than 30 seconds, advantageously of more than 60 seconds, most preferably of more than 200 seconds, said clotting time being measured in the apparatus xe2x80x9cBFT IIxe2x80x9d of DADE BEHRING (Germany) at 37xc2x0 C. This apparatus operates according to the opto-mechanical measuring principle (measure of a turbidity). A light beam passes through a plastic cuvette containing 0.5 ml of the solution to be analysed, onto a photodetector. The change of intensity of the transmitted light is converted into an electric signal. A stir bar is placed in the cuvette or cup so as to ensure homogeneity of the solution placed in the cuvette or cup.
The amount of calcium inhibiting or blocking agent can be determined by an individual skilled in the art by successive tests for adding different amounts of calcium or blocking agent.
Advantageously, the lyophilization step is carried out at a temperature of less than 40xc2x0 C. and at a pressure of less than 0.4xc3x97105 Pa. For the lyophilization, it can be worthwhile to add some specific additive(s), such as glycerol, fatty acid, etc.
The lyophilization is advantageously carried out at different temperatures below about xe2x88x9210xc2x0 C. For example, the lyophilization is first carried out at a temperature below than xe2x88x9240xc2x0 C., and then at a temperature from about xe2x88x9240xc2x0 C. to about xe2x88x9210xc2x0 C. According to a possible embodiment, the lyophilization is carried out at a temperature varying substantially continuously from a temperature below xe2x88x9240xc2x0 C. up to a temperature comprised between xe2x88x9240xc2x0 C. and xe2x88x9210xc2x0 C.
For example, the lyophilization is carried out in several steps, such as lowering the temperature to about xe2x88x9258xc2x0 C. and maintaining said temperature during a period of time (for example from 1 to 30 hours, advantageously from 1 to 15 hours) while creating a vacuum, then increasing the temperature from xe2x88x9258xc2x0 C. to xe2x88x9220xc2x0 C. or xe2x88x9230xc2x0 C. while maintaining the vacuum, then by maintaining the temperature of xe2x88x9220xc2x0 C. or xe2x88x9230xc2x0 C. while maintaining the vacuum (for example from 5 to 100 hours), then increasing the temperature to more than 20xc2x0 C., while maintaining the vacuum.
During the lyophilization, it is possible to adjust the temperature and/or the pressure for obtaining a substantially constant pore distribution through the thickness of the fibrin-fibrinogen layer. For example, the vacuum is lowered when the temperature is increased, i.e., the pressure is increased when the temperature is increased.
The lyophilization can also be controlled so as to adjust the residual moisture of the porous structure of the invention at the end of the lyophilization step. Advantageously, the residual moisture of the porous structure at the end of the lyophilization step is lower than about 7.5%, advantageously lower than about 2%, preferably lower than about 1%, most preferably lower than about 0.5%.
The end moisture of the porous structure of the invention, after the lyophilization step, can possibly be adjusted by a subsequent drying step.
According to an embodiment, in the process of the invention, at least a phosphate salt is added in an amount sufficient for having a Ca/P ratio from about 0.5 to about 5, preferably from about 1 to about 2. More preferably, one or more calcium phosphate salts (for example calcium orthophosphate) and calcium salts (calcium hydroxide) are added in an amount sufficient for having a Ca/P ratio from about 1.5 to about 2, preferably from about 1.67 to about 1.95.
According to an advantageous embodiment, the solution of fibrin or fibrinogen materials used in the process of the invention has a low albumin content, for example the solution contains less than about 5% by weight of albumin, with respect to the weight of fibrin or fibrinogen material.
According to another embodiment, particles having the porous structure of the invention are added to the solution of fibrinogen or fibrin material before the polymerization with at least partial cross-linking and/or during the polymerization with at least partial cross-linking.
According to a detail of a preferred embodiment, at least a compound selected from the group consisting of: processing aids (such as lubricant, plastifying agent, surfactant, viscosity reducing agent, etc.), fibers, polymers, copolymers, antibody, antimicrobial agent, agent for improving the biocompatibility of the structure, proteins, anticoagulants, anti-inflammatory compounds, compounds reducing graft rejection, living cells, cell growth inhibitors, agents stimulating endothelial cells, antibiotics, antiseptics, analgesics, antineoplastics, polypeptides, protease inhibitors, vitamins, cytokine, cytotoxins, minerals, interferons, hormones, polysaccharides, genetic materials, proteins promoting or stimulating the growth and/or attachment of endothelial cells on the cross-linked fibrin, growth factors, growth factors for heparin bond, substances against cholesterol, pain killers, collagen, osteoblasts, drugs, etc. and mixtures thereof is added to the solution before the polymerization with at least partial cross-linking, and/or during the polymerization with at least partial cross-linking, and/or after the polymerization with at least partial cross-linking, and/or before the lyophilization. According to another embodiment, after the lyophilzation or a partial lyophilization, the at least partly cross-linked fibrin or fibrinogen material is mixed with a compound selected from the group consisting of: additive (for example comprised within the wall of the channels or cells of the structure) or is provided with a layer containing at least an additive, said additive being selected from the group consisting of processing aids (such as lubricant, plastifying agent, surfactant, viscosity reducing agent, etc.), fibers, polymers, copolymers, antibody, antimicrobial agent, agent for improving the biocompatibility of the structure, proteins, anticoagulants, anti-inflammatory compounds, compounds reducing graft rejection, living cells, cell growth inhibitors, agents stimulating endothelial cells, antibiotics, antiseptics, analgesics, antineoplastics, polypeptides, protease inhibitors, vitamins, cytokine, cytotoxins, minerals, interferons, hormones, polysaccharides, genetic materials, proteins promoting or stimulating the growth and/or attachment of endothelial cells on the cross-linked fibrin, growth factors, cell growth factors, growth factors for heparin bond, substances against cholesterol, pain killers, collagen, osteoblasts, chondroblasts, chondrocytes, osteoclasts, hematpoeitic cells, stromal cells, osteoprogenitor cells, drugs, anti coagulants, poly DL lactate, alginate, recombinant material, triglycerides, fatty acids, C2-C24 fatty acids, drugs, etc. and mixtures thereof.
It has been observed that the use of different calcium inhibiting agents have different effects on the volume variation of the structure during the hydration, and it is possible to use inhibiting agents with a positive volume variation during the hydration (volume increase of the structure) and inhibiting agents with a negative volume variation during the hydration (volume decrease of the structure) in amounts sufficient for obtaining a structure with substantially no volume variation during the hydration.
According to a specific detail of an embodiment of the process, after the partial polymerization with at least partial cross-linking of the fibrin or fibrinogen materials, but before the lyophilization step, the polymerized material (that advantageously forms a hydrogel) is submitted to a treatment (advantageously a mechanical treatment, such as a centrifugation) for removing part of excess water present in the material. The treatment is preferably a treatment not altering the chemical links of the polymerized, partly cross-linked material.
In the process of the invention, the fibrin or fibrinogen material is polymerized with a partial cross-linking. For example, the partial cross-linking is a cross-linking of less than about 50% by weight of the fibrin molecules, more preferably from about 0.5 to about 40% by weight of the fibrin molecules, preferably from about 1 to about 20% by weight of the fibrin molecules.
The fact that the fibrin or fibrinogen material is polymerized with a partial cross-linking can be observed by an electropheris SDS-polyacrilamide gel by the absence of the xcex3-band detectable in the Fibrin-fibrinogen starting material and by the intensity of the xcex3-xcex3 band of the material after polymerization. The electrophoresis of reduced protein using 4-15% gradient polyacrilamide gels showed that for fibrin with complete cross-linking, there is no residual monomeric xcex3 chains remaining and the cross linked fibrin showed characteristic xcex2 and xcex3xcex3 polymer chains as prominent (substantially no xcex1 chain is remaining).
The intensity of the xcex3-xcex3 band is correlated to the cross-linking rate. It means that, when the intensity of the xcex3-xcex3 band is low, the cross-linking rate of the fibrin or fibrinogen is low. In order to determine the cross-linking rate in the process of the invention, solutions with different fibrinogen content were cross-linked not in presence of calcium inhibiting agent and the intensity of the xcex3-xcex3 band of the cross-linked fibrinogen solutions is determined by SDS analysis for having a reference. The intensity of the xcex3-xcex3 band of the fibrinogen polymerized in presence of the calcium blocking agent is then compared with the reference, so as to determine the amount of cross-linked fibrinogen, and therefore the percent of cross-linked fibrinogen and the percent of polymerized (not cross-linked) fibrinogen. The amount of cross-linked fibrinogen in the structure of the invention is considered as being the amount of fibrinogen cross-linked not in presence of a calcium blocking agent (in a reference solution) having substantially the same intensity for the xcex3-xcex3 band.
The lyophilization step of the solution can be carried out after placing the solution in a mould so as to give a shape to the lyophilized product. It is also possible to adjust the desired shape of the lyophilized product after lyophilization and/or after hydration thereof.
The fibrin-fibrinogen material used in the process of the invention can possibly be a recombinant fibrin-fibrinogen or a mixture of a recombinant fibrin-fibrinogen with a natural fibrin-fibrinogen material. The thrombin used can be thrombin of natural origin, recombinant thrombin, or mixture thereof.
The polymerization, with advantageously partial cross-linking, of the fibrin-fibrinogen material can possibly be carried out in presence of collagen, recombinant collagen, fibronectin, recombinant fibronectin, albumin, recombinant albumin, factor XIII, recombinant factor XIII, interactive biomaterials (such as RGD collagens) and mixtures thereof. The collagen is, for example, a photo activateable or light activateable recombinant collagen.
The thrombin is, for example, a cage thrombin, a light activateable thrombin, a recombinant thrombin, etc. According to a preferred embodiment, the thrombin is mixed with a calcium inhibiting or blocking agent, especially a anticoagulant.
It is also possible to add and mix collagen, recombinant collagen, fibronectin, recombinant fibronectin, albumin, recombinant albumin, factor XIII, recombinant factor XIII, interactive biomaterials (such as RGD collagens) and/or mixtures thereof to the hydrogel of fibrin-fibrinogen before its lyophilization.
Specific processes are:
formation of a hydrogel of fibrin-fibrinogen material according to the process of the invention, addition of bone chips to the hydrogel, lyophilization;
formation of a hydrogel of fibrin-fibrinogen material according to the process of the invention, addition and mixing of collagen (preferably photo activable), lyophilization;
formation of a hydrogel of fibrin-fibrinogen material according to the process of the invention, addition and mixing of collagen (preferably photo activable) and bone chips, lyophilization;
formation of a hydrogel of fibrin-fibrinogen material according to the process of the invention, addition and mixing of a hydrogel of collagen and preferably of bone chips, lyophilization.
The hydrogel prepared according to the process of the invention, possibly after a first treatment for removing an excess of water, can be spray-freeze dried, sprayed in drops which are thereafter freeze dried, freeze IR dried, so as to prepare particles having the structure of the invention, for example substantially spherical particles having a particle size or diameter of less than 2 mm, for example a particle size from about 500 xcexcm to about 1 mm. The particle size distribution can be adjusted as required by adjusting the parameter of the spray-freeze drying and/or the spraying.
The invention relates also to a support, advantageously a metal support, preferably a titanium containing support provided with a layer having the structure of the invention or a layer comprising particles having the structure of the invention. The support is possibly porous.
Such a support can be prepared, for example, by one of the following processes:
In a first process, the support is contacted with the solution containing the fibrin or fibrinogen material and the calcium inhibiting or blocking agent as disclosed in the process of the invention. Thereafter, the support in contact with the solution is lyophilized so as to obtain a support (for example a titanium containing support) coated with a porous structure as disclosed in the porous structure of the invention. When the support is porous, pores of the support can be filled with a product having the structure of the invention.
In a second possible process, particles having the porous structure of the invention are sprayed on a support (for example a titanium containing support) coated with an adhesion layer, for example a fibrin based layer.
In a third possible process, particles having the porous structure of the invention have been mixed with an adhesive composition, for example a biocompatible glue, such as a fibrin glue, and the adhesive composition with the particles is used for coating the support, preferably a titanium containing support (for example by spaying the composition or by brushing the composition on the support).
According to still a further possible process, lyophilized particles having the structure of the invention are mixed with thrombin and/or calcium containing compound, the mixture possibly mixed with an adhesive or liquid glue being used for coating the titanium containing support (for example provided with a glue layer). The thrombin used is for example an inactivated thrombin, for example a thrombin that is light-activated.
A further object of the invention is therefore a mixture of particles of fibrin and/or fibrinogen materials with inactivated thrombin, said mixture possibly containing further additives, such as drugs, anti-inflammatory compounds, compounds reducing graft rejection, living cells, cell growth inhibitors, agents stimulating endothelial cells, antibiotics, antiseptics, analgesics, antineoplastics, polypeptides, protease inhibitors, vitamins, cytokine, cytotoxins, minerals, proteins, interferons, hormones, polysaccharides, genetic materials, proteins promoting or stimulating the growth and/or attachment of endothelial cells on the cross-linked fibrin, growth factors, cell growth factors, growth factors for heparin bond, substances against cholesterol, pain killers, collagen, osteoblasts, chondroblasts, chondrocytes, osteoclasts, hematpoeitic cells, stromal cells, osteoprogenitor cells, drugs, anti coagulants, poly DL lactate, alginate, recombinant material, triglycerides, fatty acids, C12-C24 fatty acids, drugs, pain killers, etc., and mixtures of said compounds or a solution containing at least one of said compound.
The structure of the invention is advantageously provided with cells.
The invention relates also to a kit suitable for forming a bone substitute, said kit comprising a first chamber containing a product having the structure of the invention, and a second chamber containing an aqueous solution containing at least BMP (bone morphology proteins). The product having the structure of the invention is hydrated with the solution before its use as a bone substitute.
For example, the kit comprises a blister or chamber and a part equipped with a luer lock system to be fixed to syringe.